Evaluation of mitochondrial DNA coding region assays for increased discrimination in forensic analysis

There is an increasing trend to use mitochondrial DNA (mtDNA) analysis in criminal investigations where only limited amounts of DNA are available. However, analysis of the mtDNA control region has the drawback of low discrimination power, due to the lack of recombination that results from uniparental (maternal) inheritance. As a strategy to increase discrimination, a number of typing assays detecting variation in the mitochondrial coding region have been developed. In this study, several of these assays are evaluated for their discriminatory capacity using data obtained from 495 complete Caucasian mtDNA sequences. In order to add a local geographic perspective to this evaluation, we have also sequenced and analysed the entire mtDNA from 20 individuals of Swedish origin. We find that the coding region assays are very useful for resolving sequences with identical HVI/HVII regions. The best-performing coding region assay was able to discriminate 46% of the resolvable sequences, compared to 20–30% for the other coding region assays we evaluated.     

Sequence polymorphisms of the mitochondrial DNA control region and phylogenetic analysis of mtDNA lineages in the Japanese population

Sequence polymorphisms of the hypervariable mitochondrial DNA (mtDNA) regions HVI and HVII, and coding region polymorphisms were investigated in 211 unrelated individuals from the Japanese population. Sequence comparison of the HVI and HVII regions led to the identification of 169 mitochondrial haplotypes defined by 147 variable positions. Among them 145 types were observed in only 1 individual; the other 24 types were shared by 2 or more individuals. The gene diversity was estimated at 0.9961, and the probability of two randomly selected individuals from the population having identical mtDNA types was 0.86%. We also established phylogenetic haplogroups in the Japanese population based on the coding and control region polymorphisms and compared the haplotypes with those in other Japanese, Korean and Chinese populations. As a result, three new subhaplogroups, G4a, G4b, and N9b, and several haplotypes specific for the Japanese and Korean populations were identified. The present database can be used not only for personal identification but also as an aid for geographic or phenotype (race) estimation in forensic casework in Japan.Electronic Supplementary Material Supplementary material is available for this article if you access the article at     

Sequence polymorphism of the mitochondrial DNA control region in the Slovenian population

The forensic application of mitochondrial DNA (mtDNA) typing requires large and regionally well-defined databases. To expand the database for forensic identification purposes in Slovenia, the mtDNA control region sequences of the hypervariable regions HVI and HVII were determined in a population of 129 maternally unrelated Slovenians, using a fluorescent-based capillary electrophoresis sequencing method. A total of 111 different haplotypes resulting from 124 polymorphic positions (80 polymorphic positions in HVI and 44 in HVII) were found. Of these, 101 mtDNA types were unique, 6 haplotypes were shared by 2 individuals, 1 haplotype by 3 individuals, 2 haplotypes by 4 individuals, and the most common haplotype was found in 5 individuals. The most frequent haplotypes in the Slovenian population ,263(G), 315.1(C) and 263(G), 309.1(C), 315.1(C) are also the most common in other European populations. The data support the concept that these haplotypes may represent a common European mtDNA sequence types. The sequence poymorphisms were compared to the databases of west Austria and central Italy and the HVI and HVII sequence matching probabilities within and between populations were calculated. It is 1.1–4.5 times more likely to find a sequence match in a random pair of Slovenians than in a random Slovenian-Italian pair and in a random Slovenian-Austrian pair. The length heteroplasmy in the homopolymeric C-stretch regions located at nucleotide positions 16184–16193 in HVI and at positions 303–315 in HVII was observed in 17% and 8% of individuals, respectively. A statistical estimate of the results for this population showed the random match probability and the genetic diversity of 1.16% and 0.996, respectively.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00414-003-0394-3     

Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers

Urine samples from 20 male volunteers of European Caucasian origin were stored at 4°C over a 4-month period in order to compare the identification potential of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) markers. The amount of nDNA recovered from urines dramatically declined over time. Consequently, nDNA likelihood ratios (LRs) greater than 1,000 were obtained for 100, 70 and 55% of the urines analysed after 6, 60 and 120 days, respectively. For the mtDNA, HVI and HVII sequences were obtained for all samples tested, whatever the period considered. Nevertheless, the highest mtDNA LR of 435 was relatively low compared to its nDNA equivalent. Indeed, LRs obtained with only three nDNA loci could easily exceed this value and are quite easier to obtain. Overall, the joint use of nDNA and mtDNA markers enabled the 20 urine samples to be identified, even after the 4-month period. Electronic supplementary material Supplementary material is available for this article at .     

Helena,the hidden beauty: Resolving the most common West Eurasian mtDNA control region haplotype by massively parallel sequencing an Italian population sample

The analysis of mitochondrial (mt)DNA is a powerful tool in forensic genetics when nuclear markers fail to give results or maternal relatedness is investigated. The mtDNA control region (CR) contains highly condensed variation and is therefore routinely typed. Some samples exhibit an identical haplotype in this restricted range. Thus, they convey only weak evidence in forensic queries and limited phylogenetic information. However, a CR match does not imply that also the mtDNA coding regions are identical or samples belong to the same phylogenetic lineage. This is especially the case for the most frequent West Eurasian CR haplotype 263G 315.1C 16519C, which is observed in various clades within haplogroup H and occurs at a frequency of 3–4% in many European populations.In this study, we investigated the power of massively parallel complete mtGenome sequencing in 29 Italian samples displaying the most common West Eurasian CR haplotype – and found an unexpected high diversity. Twenty-eight different haplotypes falling into 19 described sub-clades of haplogroup H were revealed in the samples with identical CR sequences. This study demonstrates the benefit of complete mtGenome sequencing for forensic applications to enforce maximum discrimination, more comprehensive heteroplasmy detection, as well as highest phylogenetic resolution.     

Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia

This paper describes molecular genetic identification of one third of the skeletal remains of 88 victims of postwar (June 1945) killings found in the Konfin I mass grave in Slovenia. Living relatives were traced for 36 victims. We analyzed 84 right femurs and compared their genetic profiles to the genetic material of living relatives. We cleaned the bones, removed surface contamination, and ground the bones into powder. Prior to DNA isolation using Biorobot EZ1 (Qiagen), the powder was decalcified. The nuclear DNA of the samples was quantified using the real-time polymerase chain reaction method. We extracted 0.8 to 100 ng DNA/g of bone powder from 82 bones. Autosomal genetic profiles and Y-chromosome haplotypes were obtained from 98% of the bones, and mitochondrial DNA (mtDNA) haplotypes from 95% of the bones for the HVI region and from 98% of the bones for the HVII region. Genetic profiles of the nuclear and mtDNA were determined for reference persons. For traceability in the event of contamination, we created an elimination database including genetic profiles of the nuclear and mtDNA of all persons that had been in contact with the skeletal remains. When comparing genetic profiles, we matched 28 of the 84 bones analyzed with living relatives (brothers, sisters, sons, daughters, nephews, or cousins). The statistical analyses showed a high confidence of correct identification for all 28 victims in the Konfin I mass grave (posterior probability ranged from 99.9% to more than 99.999999%).     

Forensic evaluation of mtDNA in a population from south west Switzerland

The polymorphism of the two hypervariable segments (HVI and HVII) of the control region of mtDNA was analyzed in a population of 154 unrelated individuals from south west Switzerland using a fluorescent based capillary electrophoresis sequencing method. In our population data of 154 random individuals, 137 mtDNA types were observed. Of these, 124 sequences were observed only in one individual whereas 10 sequences were observed in 2 individuals, 2 sequences in 3 individuals and 1 sequence in 4 individuals. The probability of two unrelated individuals having the same sequence was 0.84%. The results were compared with four other Caucasian populations. Furthermore, the usefulness of the mtDNA sequencing was tested, for exclusion and inclusion, in 18 forensic cases including 69 evidence samples and 44 reference samples. Despite the fact that 55% of the evidence samples yielded a negative result for the nuclear DNA with the human dot quantitation system, the success rate of the mtDNA sequencing was 71.0%. This validation study proves the great usefulness and sensitivity of the mtDNA sequencing technique using nested PCR and fluorescent capillary electrophoresis.     

Quantification of mtDNA mixtures in forensic evidence material using pyrosequencing

Analysis of mtDNA variation using Sanger sequencing does not allow accurate quantification of the components of mtDNA mixtures. An alternative method to determine the specific mixture ratios in samples displaying heteroplasmy, consisting of DNA contributions from several individuals, or containing contamination would therefore be valuable. A novel quantification system for mtDNA mixture analysis has been developed based on pyrosequencing technology, in which the linear relationship between incorporated nucleotides and released light allows quantification of the components of a sample. Within five polymerase chain reaction fragments, seven variable positions in the mtDNA control and coding region were evaluated using this quantification analysis. For all single nucleotide polymorphisms quantified in this study, a linear relationship was observed between the measured and expected mixture ratios. This mtDNA quantification assay is an easy to use, fast and accurate quantification system, with the ability to resolve and interpret major and minor mtDNA components in forensic mixture samples.H. Andréasson and M. Nilsson have contributed equally to the work.     

Identification of West Eurasian mitochondrial haplogroups by mtDNA SNP screening: Results of the 2006–2007 EDNAP collaborative exercise

The European DNA Profiling (EDNAP) Group performed a collaborative exercise on a mitochondrial (mt) DNA screening assay that targeted 16 nucleotide positions in the coding region and allowed for the discrimination of major west Eurasian mtDNA haplogroups. The purpose of the exercise was to evaluate the stability and reproducibility of the self-developed multiplex-PCR and multiplex-single base extension kit by blind-testing saliva and hair shaft samples provided by the organizing laboratory. The overall success rate in obtaining useful results was high given that some of the participating laboratories had no previous experience with the technology and/or mtDNA analysis. The results of this collaborative exercise stimulate the expansion of screening methods in forensic laboratories to increase efficiency and performance of mtDNA typing, and thus demonstrates that mtDNA SNP typing is a powerful tool for forensic casework analysis.     

HVI and HVII mitochondrial DNA data in Apaches and Navajos

Most mtDNA studies on Native Americans have concentrated on hypervariable region I (HVI) sequence data. Mitochondrial DNA haplotype data from hypervariable regions I and II (HVI and HVII) have been compiled from Apaches (N=180) and Navajos (N=146). The inclusion of HVII data increases the amount of information that can be obtained from low diversity population groups. Less mtDNA variation was observed in the Apaches and Navajos than in major population groups. The majority of the mtDNA sequences were observed more than once; only 17.8% (32/180) of the Apache sequences and 25.8% of the Navajo sequences were observed once. Most of the haplotypes in Apaches and Navajos fall into the A and B haplogroups. Although a limited number of haplogroups were observed, both sample populations exhibit sufficient variation for forensic mtDNA typing. Genetic diversity was 0.930 in the Apache sample and 0.963 in the Navajo sample. The random match probability was 7.48% in the Apache sample and 4.40% in the Navajo sample. The average number of nucleotide differences between individuals in a database is 9.0 in the Navajo sample and 7.7 in the Apache sample. The data demonstrate that mtDNA sequencing can be informative in forensic cases where Native American population data are used.     

Coding region mitochondrial DNA SNPs: Targeting East Asian and Native American haplogroups

We have developed a single PCR multiplex SNaPshot reaction that consists of 32 coding region SNPs that allows (i) increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and (ii) haplogroup assignments of mtDNA profiles in both human population studies (e.g. anthropological) and medical research. The selected SNPs target the East Asian phylogeny, including its Native American derived branches. We have validated this multiplex assay by genotyping a sample of East Asians (Taiwanese) and Native Americans (Argentineans). In addition to the coding SNP typing, we have sequenced the complete control region for the same samples. The genotyping results (control region plus SNaPshot profiles) are in good agreement with previous human population genetic studies (based on e.g. complete sequencing) and the known mtDNA phylogeny. We observe that the SNaPshot method is reliable, rapid, and cost effective in comparison with other techniques of multiplex SNP genotyping. We discuss the advantages of our SNP genotyping selection with respect to previous attempts, and we highlight the importance of using the known mtDNA phylogeny as a framework for SNP profile interpretation and as a tool to minimize genotyping errors.     

Phylogenetic classification of Japanese mtDNA assisted by complete mitochondrial DNA sequences

We investigated control and coding region polymorphisms in mitochondrial DNA (mtDNA) in 100 unrelated individuals from a Japanese population and determined the basal phylogenetic haplogroup lineages in all samples under updated information. Many of the basal phylogenetic haplogroup lineages assigned on East Asian mtDNA haplogroups corresponded to those previously established. However, new haplogroup lineages such as M7a2a, M7a2b, M7a2*, M7c1b, M11b2*, G2b*, D4c1b1a, D4g2b, A4*, A9, N9b*, B4d1, B4d2, and F1e were identified and established by complete sequencing. Although sequence comparison of the 1.15-kb control region identified 84 mitochondrial haplotypes, examination of coding region polymorphisms increased the total number of haplotypes to 91. Determination of the basal haplogroup lineages increased the discrimination power of mtDNA polymorphisms for personal identification and their usefulness in determining geographic origin in forensic casework in Japanese and other East Asian populations.     

Specificity of mtDNA-directed PCR—influence of NUclear MTDNA insertion (NUMT) contamination in routine samples and techniques

Nuclear mitochondrial insertions (NUMTs) are sequences homologous to mtDNA, which are present throughout the human nuclear genome. The possibility that these sequences may be accidentally amplified in reactions directed to mtDNA has been raised and evaluated by different groups and by different means. Despite that, data is still missing on the specificity of PCRs in routine procedures in what concerns contamination with nuclear mtDNA insertions (NUMTs). In this work, we performed PCR sequencing reactions with primers directed either to mitochondrial or to NUMT DNA with different annealing temperatures and in different tissues. We observed that (a) contamination with NUMTs depends on the sample and tissue, and (b) employing routine techniques, there is no risk of co-amplification. Only when mtDNA is almost completely removed from the samples does the number of NUMT copies exceed mitochondrial sequences, i.e., only in samples with virtually no mtDNA, such as those resulting from preferential semen lysis, is there a risk of accidental amplification of NUMTs. We suggest that to evaluate a possible co-amplification of NUMT DNA, it is more relevant to take into account sample processing and original tissue of the samples, and consequently the relative proportions of NUMT and mtDNA, rather than the presence of NUMTs by itself, irrespectively of its proportion.     

Successful nuclear DNA profiling of rootless hair shafts: a novel approach

Historically, rootless hair shaft samples submitted to a forensic laboratory for DNA analysis are reserved for mitochondrial DNA (mtDNA) analysis due to the presence of highly degraded as well as insufficient amounts of nuclear DNA. Although mtDNA has been very successful in obtaining results from rootless hair, this system has its limitations, namely, it is a lineage marker that cannot differentiate between maternally related genotypes. Given the high incidence of hairs as forensic evidence, there is a need for the use of a nuclear DNA test system capable of producing reliable results for hair shaft forensic evidence. This study reports the utilization of an enhanced DNA extraction methodology for hairs, in combination with a recently developed novel, nuclear DNA typing assay, InnoTyper® 21, to improve the success rate for obtaining informative results from highly compromised, degraded, and trace forensic samples such as rootless hair shafts. The InnoTyper 21 kit is a small amplicon retrotransposon marker typing system compatible with currently used capillary electrophoresis platforms. This system contains 20 Alu element markers, ranging in size from 60 to 125 bp, making the assay highly sensitive for extremely degraded forensic samples and thus enabling recovery of nuclear DNA profiles from samples that would otherwise require mtDNA sequencing. A subset of samples was also tested with the GlobalFiler kit with less success due to the larger amplicon sizes in comparison with InnoTyper 21. Results were variable but very promising, with approximately 40% of the total number of hairs tested producing interpretable nuclear DNA profiles with InnoTyper 21. These results demonstrate the ability of the utilized methodologies to produce nuclear DNA results with high statistical power from rootless hair shafts.

     

DNA typing from human faeces

A method has been developed for the forensic analysis of faeces by DNA amplification and direct sequencing of a polymorphic segment of mitochondrial DNA. Starting from as little as 10 mg wet weight of faeces, DNA was extracted by a variety of protocols and amplified using primers specific to hypervariable region I of the mitochondrial control region. The resulting amplification products were sequenced in solid phase using an automated DNA sequencer. In total, mtDNA sequences were generated from the faeces of nine Caucasians and compared with sequences generated from their respective blood samples. Sequences of faeces and blood samples from the same individual were identical in every case, but a range of 1–10 nucleotide differences was observed between individuals, with an average sequence variation of approximately 4.88 per 400 bp. Of the various extraction protocols assessed in this study, greatest success rates were achieved using magnetisable beads to bind and purify the DNA. STR analysis of DNA extracted from faeces was not routinely possible.     

Validation of the PLEX-IDTM mass spectrometry mitochondrial DNA assay

For very challenged biological samples, mitochondrial DNA (mtDNA) analysis can often provide results when the more traditional nuclear DNA markers fail. While reliable, the current method of mtDNA analysis by Sanger sequencing is expensive, labor-intensive, and time-consuming and is limited by its inability to quantify mixed samples. The Abbott PLEX-ID? instrument, which enables analysis of mtDNA amplicons via electrospray ionization mass spectrometry (ESI-MS), produces comparable accuracy and sensitivity while offering a faster and less expensive alternative to Sanger sequencing. Unlike Sanger sequencing, this system is capable of quantifying DNA species and thus may be exploited for evaluating heteroplasmy and, possibly, mixture deconvolution. Validation studies of the PLEX-ID? mtDNA assay confirmed that the instrument is highly sensitive and capable of yielding reproducible results. Samples commonly encountered in a forensic setting, as well as population samples, were typed correctly. The PLEX-ID? mtDNA assay yields reliable results for single-source samples, which are the same sample types currently examined in forensic laboratories via Sanger sequencing, at a level that meets or exceeds that of the current method. While the instrument has the demonstrated capability to quantify mixed samples, the specific assay design for mtDNA analysis can be used only in a limited fashion to analyze mixtures due to the formation of chimeric mtDNA products.     

A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation.     

A rapid mtDNA assay of 22 SNPs in one multiplex reaction increases the power of forensic testing in European Caucasians

We have developed a multiplex mitochondrial (mtDNA) assay of 21 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable region 1 (HVR1) and hypervariable region 2 (HVR2) that can be amplified in a single reverse touchdown polymerase chain reaction. Single base extension using the SNaPshot technique is also carried out as one multiplex. Besides the nine major European haplogroups (i.e. H, I, J, K, T, U, V, W, and X), 16 additional subclades (i.e. N1, X2, X2b, U2′-4/7′-9′, J/T, J1, J1c, HV, H1, H1a1, H1c, H3, H4, H6a, H7a H10) can be detected and classified into a phylogenetic mtDNA tree. By analyzing 130 Caucasoid samples from Germany, 36 different haplotypes were found resulting in a power of discrimination of 93.2%. Although 49% of all samples belonged to superhaplogroup H, the most common haplotype, i.e., haplogroup-specific SNPs plus haplogroup unspecific SNPs, had a frequency of only 18%. This assay is applicable for high-throughput mtDNA analysis and forensic mass screening. It will give additional information to the common control region sequencing of HVR1 and HVR2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of single nucleotide polymorphisms within the mtDNA genome of the domestic dog to discriminate individuals with common HVI haplotypes

We sequenced the entire ～16 kb canine mitochondrial genome (mtGenome) of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. We then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. Haplotype frequencies in our data set were highly correlated with previous ones (e.g., F(ST)=0.02, r=0.90), suggesting the total data set reasonably reflected the broader dog population. A total of 128 HVI haplotypes was represented. The 10 most common HVI haplotypes (n=184 dogs) represented 53.3% of the sample. We identified a total 71 SNPs in the mtGenomes (external to the control region) that resolved the 10 most common HVI haplotypes into 63 mtGenome subhaplotypes. The random match probability of the dataset based solely on the HVI sequence was 4%, whereas the random match probability of the mtGenome subhaplotypes was <1%. Thus, the panel of 71 SNPs identified in this study represents a useful forensic tool to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).     

Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs,assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.